PROJECT SUMMARY Vulvodynia is a chronic pain disorder affecting up to 15% of women and resulting in substantial impairment in health related quality of life. Provoked Vestibulodynia (PVD), a form of localized provoked vulvodynia (formerly called vulvar vestibulitis syndrome) consists of severe pain upon vaginal penetration and objective findings of vulvar burning pain when touching the vulvar vestibule with a cotton swab in the absence of detectable infectious, neurological or immunological explanation The treatment of the disorder is hampered by a lack of knowledge regarding the biological mechanism underlying symptoms. The human vaginal microbiota play a key role in preventing a number of urogenital diseases. Research on the association between altered composition of the vaginal microbiome in vulvodynia is sparse, but provides tentative support for the involvement of microbiota. Discovering an association between microbiome and PVD has great implications for personalized prevention, practical, targeted diagnostic testing, and personalized therapy for girls and women with PVD. Research indicates brain regions involved in the processing/modulation of signals from the external genitals are altered in vulvodynia subjects and that these alterations correlate with subjective reports of pain and vaginal muscle tenderness. There is growing preclinical and emerging clinical evidence that the microbiota and their metabolites may play a significant role in the modulation of brain activity and central signaling systems, and a potential role in the etiology and pathophysiology of pain and psychiatric disorders. In the current proposal, we will assess the vaginal microbiota and plasma and vaginal metabolites of 50 female healthy controls and 50 PVD subjects. The PVD subjects will have also been enrolled in our extensive phenotyping study (Labus/Rapkin R01 HD076756) where we use brain imaging to assess the structure and function of the brain along with clinical, genetic, physiological, and biological parameters. This study will test the hypotheses that 1) Microbiota composition and/or metabolite profiles discriminate PVD patients from HCs as well as within PVD subjects, 2) Microbiota composition and/or metabolite profiles are correlated with PVD symptom severity, pressure pain sensitivity, perceived stress and trauma history, and 3) Microbiota composition and/or metabolite profiles are correlated with brain regions altered in PVD, suggesting a possible interaction between the microbiota and the brain structure/function.